Harnessing the Gut Microbes of Low Abundance With a Bent-Capillary-Centrifugal-Driven (BCCD) Microdroplet Method

Gut microbe cultivation is essential for studying host-microbiota interactions. Traditional cultivation methods often fail to recover microbial species at low abundance (< 0.1%). To overcome this limitation, we employed the bent-capillary-centrifugal-driven (BCCD) method to encapsulate and cultivate fecal microbes in microdroplets. Fecal bacterial cells were distributed into ~50 nL microdroplets via the BCCD generator, and the microdroplets were dispersed in the oil phase and further incubated under controlled conditions. The BCCD method significantly increased the frequency of microbes at low abundance. Compared to the plate-based method, BCCD-based cultivation produced distinct microbial community structures and exhibited significantly lower temporal variation during cultivation (p < 0.05). Lineage-specific effect size (LEfSe) analysis revealed that BCCD-based cultivation enriched 29 low-abundant bacterial genera, whereas the plate-based method enriched 26. Using this method, we isolated 1,049 bacterial strains representing 123 species and 58 genera, including 8 novel species. Among the isolated and cultivated genera, 62.1% (36/58) were microbes of low abundance in the original fecal sample, and 41.4% (12/29) of the BCCD-specific enriched genera were successfully obtained. Notably, comparison with four major gut microbial culture studies revealed 45 species were exclusively recovered in this work. Taken together, the results demonstrated that our BCCD-based cultivation method effectively enriched and facilitated the isolation and cultivation of microbes at low abundance and novel gut bacterial species.